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Process for producing physiologically active protein using genetically modified silkworm

a technology of genetically modified silkworm and protein, which is applied in the field of process for producing physiologically active protein using genetically modified silkworm, can solve the problems of difficult to extract and purify the difficulty of purifying the target protein from the body fluid of the silkworm, so as to achieve the effect of reducing the amount of time and labor required for inoculation of recombinant virus, reducing the amount of time and labor required,

Inactive Publication Date: 2010-02-09
NAT INST OF AGROBIOLOGICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]In consideration of these circumstances, the object of the present invention is to provide a genetic engineering material for insects that does not require the use of recombinant baculovirus and enables a target protein having physiological activity to be purified easily, while simultaneously providing a process for producing exogenous protein using that genetic engineering material.
[0014]As a result of extensive studies, the inventors of the present invention found that, by inserting a DNA sequence having a structure in which a gene that encodes a target protein is coupled downstream from a promoter specifically expressed in silkworm silk glands into silkworm chromosomes using DNA originating in a transposon, the target protein is produced in the silk glands, or the cocoon and the silk thread, in a form that retains physiological activity, thereby leading to completion of the present invention. In the present invention, as the recombinant protein can be recovered from the silk glands or silk and cocoon thread without containing a large amount of contaminants, it offers the advantage of allowing the target protein to be purified easily. Moreover, as a virus like baculovirus is not used, virus deactivation is not necessary thereby allowing the recombinant protein to be produced both easily and safely.
[0016]The inventors of the present invention succeeded in producing a large amount of exogenous protein in silk gland cells, outside silk gland cells and in silk thread by inserting into silk gland cells and so forth an expression gene cassette in which the DNA sequence of the 5′ terminal portion and the DNA sequence of the 3′portion of fibroin H chain gene were fused to an exogenous protein gene, and were able to establish an exogenous protein production technology that facilitates purification by producing an exogenous protein using silk glands instead of using a recombinant baculovirus.

Problems solved by technology

Although extensive research has been conducted on technologies for producing recombinant proteins using insects, there are problems such as the need to deactivate and contain the recombinant baculovirus in which the exogenous protein gene has been incorporated, or the need to take a lot of time and labor associated with inoculating the recombinant virus.
In addition, the production of exogenous protein in silkworms using recombinant baculovirus had the problem of it being difficult to extract and purify the target protein from body fluid containing large amounts of contaminating proteins.
Although studies have been conducted on technologies, for producing recombinant proteins, in which an exogenous protein gene has been inserted into silkworm chromosomes, these have problems consisting of the small amount of target exogenous protein produced and the difficulty in purifying the target protein from silkworm body fluid.

Method used

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  • Process for producing physiologically active protein using genetically modified silkworm
  • Process for producing physiologically active protein using genetically modified silkworm
  • Process for producing physiologically active protein using genetically modified silkworm

Examples

Experimental program
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example 1

Preparation of Bombyx mori Genomic DNA

[0088]Fifth instar third day silkworms were dissected to remove posterior silk gland tissue. After washing with 1×SSC, 200 μl of DNA extraction buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 100 mM NaCl) were added. After adding Proteinase K (final concentration: 20 μg / ml) and adequately grinding up the tissue with a grinder, 350 μl of DNA extraction buffer and 60 μl of 10% SDS were added followed by incubating for 2 hours at 50° C. After adding 500 μl of Tris-HCl-saturated phenol (pH 8.0) and mixing for 10 minutes, the supernatant was recovered by centrifuging for 5 minutes at 4° C. and 10,000 rpm. After adding an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) to the supernatant and mixing, the resulting mixture was centrifuged. Phenol / chloroform / isoamyl alcohol was again added followed by centrifuging and recovery of the supernatant. After adding an equal volume of chloroform / isoamyl alcohol (24:1) and mixing, the mixture wa...

example 2

Gene Preparation

[0089]The genes used were acquired by PCR by producing primers for the sequences on both ends using known sequences and using suitable DNA sources for the templates. Restriction sites were added to the ends of the primers for the subsequent gene construction procedure.

[0090]Feline interferon-ω gene (base numbers 9-593 of GenBank registration no. S62636) was acquired by PCR using two types of primers consisting of primer 3 (SEQ. ID No. 3) and primer 4 (SEQ. ID No. 4) and using baculovirus rBNV100 encoding feline interferon-ω gene for the template. rBNV100 can be produced by, for example, cutting out FeIFN gene from a plasmid extracted from E. coli(pFeIFN1) (Patent Microorganism Depository No. 1633), coupling to a silkworm cloning vector (T. Horiuchi, et al., Agric. Biol. Chem., 51, 1573-1580, 1987), and co-transfecting silkworm established cells with the recombinant plasmid produced and silkworm nuclear polyhedrosis virus DNA.

[0091]Sericin-1 gene promoter (base number...

example 3

Production of Plasmids for Gene Insertion

[0094]pigA3GFP (Nature Biotechnology 18, 81-84, 2000) was used for the plasmid for gene insertion. Namely, vector pigA3GFP is a vector in which after removing a region encoding transposase from plasmid p3E1.2 disclosed in U.S. Pat. No. 6,218,185, an A3 promoter (base numbers 1764-2595 of GenBank registration no. U49854), GFP originating in pEGFP-N1 vector (Clontech) and poly A addition sequence originating in SV40 (base numbers 659-2578 of GenBank registration no. U55762) are inserted into that portion (Nature Biotechnology 18, 81-84, 2000). The expression unit of feline interferon-ω gene was inserted at the XhoI site upstream from the A3 promoter. The expression units of the inserted genes consisted of a sericin-1 gene promoter-feline interferon-ω-bovine growth hormone poly A addition sequence (SEQ. ID No. 1), or a fibroin H chain gene promoter-feline interferon-ω-bovine growth hormone poly A addition sequence (SEQ. ID No. 2). The following ...

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Abstract

The present invention provides a genetic engineering material for insects that enables a target protein to be purified easily, without requiring the use of recombinant baculovirus, while simultaneously providing a process for producing exogenous protein using that genetic engineering material. A gene recombinant silkworm is obtained by inserting an exogenous protein gene such as a cytokine gene coupled to a promoter that functions in silk glands into a silkworm chromosome. An exogenous protein such as a cytokine is then extracted and purified from the silk glands or cocoon of that silkworm or its offspring. A large amount of exogenous protein can be produced within silk gland cells, outside silk gland cells or in silk thread or a cocoon by inserting an expression gene cassette, in which the DNA sequence of the 3′ terminal portion and the DNA sequence of the 5′ terminal portion of fibroin H chain gene are fused to the exogenous protein gene, into silk gland cells and so forth.

Description

TECHNICAL FIELD[0001]The present invention relates to a process for producing a recombinant cytokine using a silkworm incorporating a cytokine gene in its chromosomes. In addition, the present invention relates to a gene recombinant silkworm having the property of producing a recombinant cytokine in a silk gland or cocoon and silk thread, and a vector for inserting an exogenous gene into silkworm chromosomes for producing the recombinant silkworm. In addition, the present invention also relates to a process for producing exogenous protein using insect cells, insect tissue or insects to which a gene has been inserted using the aforementioned vector. Moreover, the present invention relates to silk thread containing an exogenous protein produced by a recombinant silkworm obtained in the present invention.BACKGROUND ART[0002]The production of exogenous proteins using gene recombination technology is used in various industries. The hosts used for their production consist mainly of E. col...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N15/00C12P21/00A01K67/033A01K67/04C07K14/52C12N15/85
CPCA01K67/033A01K67/0335A01K67/04C07K14/52C12N15/8509A01K2217/05A01K2267/01C12N15/09C12N15/63C12N15/10
Inventor HIRAMATSU, SHINGOTANAKA, TAKASHIYAMADA, KATSUSHIGETAMURA, TOSHIKI
Owner NAT INST OF AGROBIOLOGICAL SCI
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